TCR-mimic bispecific antibodies to target the HIV-1 reservoir

Significance Novel approaches to promote killing of HIV-1–infected cells are necessary for elimination of the latent reservoir, the main barrier to a cure. Here, we utilized a diverse phage-display library to construct T cell receptor (TCR)-mimic antibodies to HIV-1 peptide-major histocompatibility complexes (pMHC). We show that single-chain diabody forms of these antibodies recognize distinct epitopes in Gag and reverse transcriptase in a specific manner and induce T cell-mediated killing of HIV-1–infected CD4+ T cells. This study lays the groundwork for future exploration of pMHC-based immunotherapeutic approaches toward elimination of the latent reservoir once effective latency-reversing strategies are developed.


Phage panning and characterization
Phage panning was performed as previously described (1). Briefly, panning consisted of four rounds of negative and positive selection. Negative selection was performed against naked streptavidin beads, free streptavidin, and irrelevant, and biotinylated A2-pMHC pre-conjugated to streptavidin beads. Negative selection was also performed against A2-bearing cells (T2s, RPMI-6666s). Positive selection was performed for each round of panning with decreasing amounts of the relevant biotinylated monomer conjugated to streptavidin beads. After each round of panning, phage were eluted with glycine (pH 2), neutralized with Tris-HCL (pH 9), amplified in SS320 cells (Lucigen), and concentrated in PEG-NaCl. Phage eluted from the 4 th round of panning were diluted such that infection of bacteria produced single colonies. Individual bacterial colonies were inoculated in deep 96-well plates to produce monoclonal phage. Monoclonal phage supernatants were tested for their ability to bind to the target versus irrelevant pMHC using ELISA and flow cytometry. Phage supernatants were added to streptavidin ELISA plates pre-coated with biotinylated A2-monomers of the relevant or irrelevant pMHC, and phage binding was assessed using a rabbit anti-M13 antibody (Pierce) and a secondary conjugated to HRP. To assess phage binding to pMHC on the surface of cells, T2 cells were pulsed with 50 ug/ml peptide and 10 ug/ml B2M for 4 hrs to overnight at 37°C in RPMI 1640 + Glutamax with 1% Pen/Strep. Pulsed cells were washed once with PBS, incubated with monoclonal phage, and then stained with rabbit anti-M13 (Pierce) followed by a PE-conjugated donkey anti-rabbit secondary (Biolegend). Phage-stained cells were acquired on the Intellicyt flow cytometer and analyzed with FlowJo.

scDb Expression and Purification
Monoclonal phages that stained specifically for a particular peptide-pulsed target were sequenced to identify the expressed Fab. Gene blocks expressing this Fab fragment, the UCHT1 Fab fragment against CD3, and C'terminal His tag were cloned via Gibson assembly into the pcDNA3.4 vector backbone. scDb constructs were sequenced, amplified, and expressed via transfection into 293T cells. scDbs were purified using nickel columns (Capturem His-tag miniprep columns) using 400 mM imidazole and were desalted to remove imidazole using Zeba Spin columns. scDbs were run on SDS-PAGE and quantified using iBright densitometric analysis in comparison to a BSA standard. Large-scale preparations of scDbs were produced by ThermoFisher.  The mass spectrum at scan 767 shows precursor ion amplitudes for both light (30,000) and heavy (13,300) forms. 30000/13300 * 100 = 226 attomoles. Over 2.5 million cells this is a copy number of 54 per cell. C, D) As above with 2500/5850*100 = 43 attomoles or 10 copies/cell. The mass spectrum at scan 241 shows the precursor ion amplitude of the heavy form but to see the light form light form requires an expanded scale (C). That this minor peak is light ILK is shown by its Poisson signature in Fig. 1. The base isotope peak at 331.203 is overlapping with background ions, hence the first isotope peaks (marked with asterisks) is used for quantitation. 570/22400 * 200 = 5 attomoles which is less than 1 copy/cell.

Fig. S4
: Selection of scFv specific for ALT, ILK, and VLA pMHC. A) Monoclonal phage were amplified in bacteria in a 96-well plate format after 4 rounds of selection and then incubated in streptavidin ELISA plates coated with biotinylated ALT pHLA-A2, ILK pHLA-A2, VLA pHLA-A2, or an isotype pHLA-A2 control. Plates were washed and phage were detected by absorbance at 450 nm using rabbit anti-M13 and an HRP-conjugated mouse anti-rabbit secondary antibody. Arrows indicate phage whose scFv sequences were utilized to construct the HA29, HI12, HV114, and HV115 scDbs described in the manuscript. Well H12 was not inoculated with phage and therefore served as a no phage control. pMHC-specific monoclonal phage clones that bound >2 fold higher to the target vs irrelevant pMHC-A2 in monoclonal ELISAs were tested for binding on T2 (HLA-A*02:01 + ) cells pulsed with 50 ug/ml of the indicated peptide. Phage binding was assessed by flow cytometry using a rabbit anti-M13 primary antibody and a donkey anti-Rabbit secondary antibody conjugated to phycoerythrin (PE). Phage binding was quantified by PE median fluorescence intensity (MFI), and the ratio of monoclonal phage bound to cells pulsed with the cognate or irrelevant peptides plus beta-2 microglobulin (B2M) was used to rank pMHC-specific phage by specificity. A-C) The MFI ratio of HA, HI, or HV-specific phage binding to cognate (ALT, ILK, or VLA peptides, respectively) versus irrelevant peptide-pulsed cells is plotted. D) Peptide stabilization of cellsurface HLA-A2 on T2 cells is shown with all three candidate target peptides. E) Representative histograms of HA29 phage clone binding to T2 cells pulsed with 10 ug/ml B2M only (gray) or B2M + 50 ug/ml ALT (green), ILK (orange), or VLA peptides. A large shift in phage binding with ALT-pulsed cells is observed. F) Monoclonal phage clone HI12 shows increased binding to T2 cells pulsed with the cognate peptide (ILK) versus irrelevant peptides (VLA and ALT). G) Additional monoclonal phage screening via flow cytometry for VLA-specific phage yielding specific clones HV115 and HV154 are shown. Fig. S6: Selection of HA29 and HI12-scDbs for further characterization. A) T2 cells were pulsed with 1 ug/ml ALT or ILK peptides and 10 ug/ml B2M and co-cultured with pre-activated CD8 + T cells at a 1:1 E to T ratio. HA or HI-specific scDbs were added at the indicated concentrations. After 72 hrs, supernatants from co-cultures were snap frozen and assayed for secreted MIP1β. B) T2 cells were pulsed with the ALT or ILK peptides at the indicated concentrations and 10 ug/ml B2M. Pulsed T2 cells were incubated with pre-activated CD8 + T cells (1:2 E to T) and with 0.25 nM of HA29, HA122, and HI155-scDbs and .5 nM of HI12-scDb for 72 hrs. Co-culture supernatants were harvested and assayed for MIP1β. C) T2 cells were pulsed with indicated concentrations of the VLA peptide and co-cultured with pre-activated CD8 + T cells in the presence of 25 ng/ml HV115. CTL activation in response to decreasing doses of peptide was measuring using surface CD69 + levels. Figure S7: Characterization of HA29-scDb binding. We generated a library of 171 positional scanning variant peptides by substituting each residue of the original peptide with the 19 other possible amino acids as previously described (1, 2). T2 cells were then pulsed with each variant at 10 uM and co-cultured with pre-activated CD8 + T cells from healthy donors. A) Supernatants were assayed for MIP1β at 24 hours, and the mean of three technical replicates is plotted as a heatmap. Black boxes indicate the amino acids in the parental peptides. B) To assess for differential peptide affinity for A2 and peptide-induced A2 upregulation on T2 cells post-pulsing, we also stained for surface HLA-A2 levels.

Fig. S8: HA29 and HI12-scDbs induce polyfunctional CD8 + T cell responses. T2 cells were pulsed
with the ALT or ILK peptides at the indicated concentrations. 10 X 10 4 peptide-pulsed cells were cocultured with 5 X 10 4 pre-activated CD8 + T cells (1:2 E to T) in the presence of .25 nM HA29 or HI12 scDbs for 72 hrs. Supernatants from co-cultures were assayed for the indicated effector molecules using Legendplex. Specific activation of CD8 + T cells by HA29 and HI12 was observed in response to cells pulsed with the cognate but not irrelevant pMHC at doses down to the nanomolar range.

Fig. S9: HA29-scDbs induce viral suppression even in infected cells with A2 downregulation. A)
Suppression assays were set up using target cells (activated CD4 + T cells from an A2-expressing healthy donor) infected with ΔEnv NL4.3 EGFP and co-cultured with autologous pre-stimulated CD8 + T cells for 72 hrs (see Methods) in the presence of HA29 or H2-scDb. A) MFI of surface HLA-A2 (BB7.2 clone) levels are shown from GFP+ or GFP-cells from two biological replicates of infected cells used in suppression assays. B) Representative flow cytometry plots of residual viable A2+ GFP + cells after 3 days of coculture in a suppression assay, showing a dose-dependent decrease with the HA29-scDb but no decrease with the H2-scDb. C) Healthy donor HLA-A*02:01 + CD4 + T cells were activated with CD3/CD28 Dynabeads for 72 hrs and infected with ΔEnv NL4.3 EGFP. Two days post infection, infected or uninfected CD4 + T cells were co-cultured at a 1:3 E:T ratio with autologous human CD8 + T cells (15 X10 4 targets and 5 X10 4 CD8 + T cells) with the HA29-scDb at 25 ng/ml for 72 hr. scDb-induced CD8 + T cell activation was measured by MIP1β ELISAs from co-culture supernatants. D) %Viable cells as determined by flow cytometry in uninfected co-cultures to assess for background killing by increasing doses of HA29-scDb. Data in C show mean ± SD analyzed by two-way ANOVA followed by Tukey's Multiple Comparison Test. Data in D represent the mean ± SD of three independent experiments analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.